CN1211191A - Gel formulations containing growth factors - Google Patents

Abstract

Gel formulations containing polypeptide growth factors having human mitogenic or angiogenic activity are provided. The gel formulations are useful for topical or incisional wound healing for cutaneous wounds, particularly in the anterior chamber of the eye. The gel formulations also comprise a water soluble, pharmaceutically or ophthalmically compatible polymeric material for providing viscosity within various ranges determined by the application of the gel. The gel formulations provide controlled release and increased contact time of the growth factor to the wound site.

Description

Gel formulations containing growth factors

Background of the invention

This application is a continuation-in-part of U.S. Serial No. 520,798 filed August 31, 1995 which is a continuation-in-part of U.S. Serial No. 135,230 filed October 12, 1993 which was a continuation-in-part of U.S. Serial No. 135,230 filed October 12, 1992 U.S. Serial No. 974,013 dated May 20, 1991, which is a continuation-in-part of U.S. Patent No. 5,427,778, which is a continuation-in-part of now-abandoned U.S. Serial No. 703,584, filed May 20, 1991, which is a continuation-in-part of a now-abandoned U.S. Patent No. 703,584 filed May 20, 1991 Continuation-in-part of US Serial No. 233,493 dated August 19, 1988, which is a continuation-in-part of now abandoned US Serial No. 098,816 filed September 18, 1987.

In this material, reference is made to various literature, patents and patent applications. These documents, patents and applications are hereby incorporated by reference in their entirety to more fully describe the state of the art as of the dates described and claimed herein as known to those skilled in the art.

The present invention provides gel formulations of human mitogenic and angiogenic polypeptide growth factors.

Human polypeptide growth factors are molecules that regulate the growth of normal human cells. A number of human polypeptide growth factors have been identified and their chemical structures have been determined. Included in this class are: epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGF-α), transforming growth factor Beta (TGF-β), insulin-like growth factors (IGF-I and IGF-II), and nerve growth factor (NGF). Due to their ability to stimulate cell growth, human polypeptide growth factors are thought to be useful in promoting processes that promote wound healing.

To date, no suitable delivery system for growth factors for the treatment of wounds has been provided. In particular, it is desirable to find a drug delivery system that can control the release of growth factors to the wound and adhere to or remain in the wound for a relatively long time to increase the contact time of the growth factor with the wound. The present invention provides such delivery systems in the form of gels containing growth factors. The biocompatible gel material can be used for drug delivery to the injured area, and has the advantages of controlled release drug delivery and providing a moist environment for the injured area.

Summary of the invention

The invention provides an aqueous gel preparation or viscous solution for controlled release of growth factors to wounded areas. The particular formulation employed will depend on the type of application desired. Available in three applications, a gel for topical or incisional injury treatment, a gel for the treatment of anterior chamber injuries of the eye and a low viscosity aqueous formulation for more fluid formulations requiring a higher water content Applications.

Aqueous gel formulations for topical or incisional injury treatment comprising a therapeutically effective amount of a polypeptide growth factor having human mitogenic or angiogenic activity. Additionally, the formulation contains a water-soluble, pharmaceutically acceptable polymeric material to provide a viscosity in the range of 1,000 to 12,000,000 cps. Viscosity measurements are usually made at room temperature or at elevated temperature. Aqueous gel formulations for the treatment of lesions in the anterior chamber of the eye include water soluble ophthalmically compatible polymeric materials to provide viscosities in the range of 1,000 to 100,000 cps at room temperature. Low viscosity aqueous formulations include water soluble pharmaceutically acceptable or ophthalmically compatible polymeric materials to provide viscosities in the range of 1 to 5,000 cps at room temperature. Formulations of low viscosity are preferred for the treatment of eye injuries. However, it can also be used in the treatment of other types of wounds, especially when used to moisten bandages placed on wounds.

The gel formulations of the present invention have the advantage of being adherent to wounds and conforming to irregular body or wound contours. The gel can be applied directly to the wound or together with an adaptable porous or microporous substrate, eg in the form of a coating. Further, the gel also has the advantages of high water content (which can keep the wound moist), absorbs wound exudate, is convenient to apply to the affected area and is conveniently removed by washing. The gel has a cooling sensation when applied to the wound, thereby increasing patient comfort and acceptance of the preparation, especially on sensitive wounds.

The invention relates to a system for controlled release and administration of growth factors in gel formulations to affected areas. Controlled-release dosing refers to drug release at levels sufficient to maintain therapy over a period of up to 24 hours or more. It has been reported that prolonged exposure of growth factors to the wound site is necessary to obtain a significantly increased rate of wound healing. The gel formulations of the present invention increase the contact time of the growth factors on the lesion and provide a sustained release dosage form. This is an important advantage, as it allows less frequent administration of the affected area and thus less disturbance of the affected area and its cellular components, especially at different phases of mitosis.

Detailed description of the invention

The aqueous gels of the present invention have different viscosities depending on the use of the gel. Viscosity is a measure of a liquid's resistance to flow. is defined as the ratio of shear stress to shear rate. Shear stress is the resistance of a liquid to flow under the influence of an external force, that is, the resistance of molecules in the body to an external force. Shear stress is defined as the ratio of force to applied area. When a shear force is applied to a liquid, assuming a laminar fluid, the layers of the liquid flow at different velocities. The relative velocity of the flow of the layers is only one factor of the shear rate. The other is the distance, or gap between the shear planes. Thus, shear rate is defined as the ratio of gel viscosity to gap. The dimension of viscosity is dyne/second per square centimeter. These dimensions are called poises. The dimension of viscosity involved in this article is centipoise (cps) unless otherwise specified, measured by a Brookfield viscometer. Unless otherwise specified, all viscosity values are at room temperature, such as 22-25°C.

The polypeptide growth factors involved in this paper have human mitogenic or angiogenic activity, selected from the group consisting of EGF, acidic FGF, basic FGF, PDGF, TGF-α, TGF-β, angiopoietin, NGF, IGF-I, Group of IGF-II or mixtures thereof. It is contemplated that biologically active fragments or chemically synthesized derivatives of these growth factors may be used rather than the entire naturally occurring molecule. In addition to mitogenic activity, EGF, FGF family, TGF family and angiogenin have been reported to have angiogenic activity. Growth factors are preferably produced by recombinant DNA techniques.

As used herein, human EGF refers to EGF having the polypeptide sequence proposed by Urdea, M.S. et al., Proc. National Academy of Sciences USA, 80:7461-7465 (1983), or a substantial portion thereof. Human EGF also refers to any naturally occurring variants of human EGF such as γ-urogastrin, epidermal growth factor, human epidermal growth factor and other growth factors that can be isolated from natural resources, produced by DNA recombinant technology or chemically synthesized.

The EGF used in the present application includes biological activity similar to that shown by natural human EGF polypeptide and has the same biological activity as determined by recognized biological assays such as the EGF receptor binding assay described in U.S. Patent No. 4,717,717 and has , its receptors and related proteins"Experimental Cell Research, 164:1-10 (1986) Discussed conserved amino acid residues and common disulfide bond position types of polypeptides. Thus, EGF includes EGF produced by recombinant DNA technology, mouse EGF isolated from mouse submandibular gland, rat EGF, and natural human epidermal growth factor, which can be isolated from human urine, and any of the aforementioned biologically active Derivatives and related polypeptides, including precursors of epidermal growth factor that are converted to active epidermal growth factor by proteolytic enzymatic processing.

PDGF is the main mitogen in serum, which promotes the proliferation of cells of mesenchymal origin, such as fibroblasts, glial cells and smooth muscle cells in vitro. Amino acid sequence data indicate that PDGF consists of two distinct but homologous polypeptide chains, an A-chain and a B-chain. These two chains have been found to be dimers of PDGF-AA, PDGF-B and PDGF-AB. The amino acid sequences of A chain and B chain of PDGF have been determined, and the amino acid sequence of B chain was proposed by Johnsson, A et al., 1984, EMBO Journal, 3:921-928. The phrase "rhPDGF-B" as used herein means the PDGF human BB homodimer produced by recombinant DNA techniques.

A therapeutically effective amount of polypeptide growth factors used in the present invention may range from about 0.01 to 1000 micrograms/ml. Preferably the growth factor concentration is about 1-500 microgram/ml, more preferably 1-100 microgram/ml. The gel of the invention can continuously release the polypeptide growth factor.

The gel-forming material of the present invention may be a water-soluble polymer capable of forming a viscous aqueous solution or a water-insoluble, water-swellable polymer such as collagen, which may also form a viscous solution. Swellable polymers are polymers that absorb water rather than dissolve in it. The cross-linked forms of the polymers described herein may also not be water-soluble, but water-swellable. Therefore, polymers in a crosslinked form belong to the scope of the present invention. Crosslinking involves covalently bonded polymer chains and bifunctional reagents such as glutaraldehyde. Those skilled in the art will appreciate that certain polymers may also be used in salt or partially neutralized form to render them water soluble. For example, sodium hyaluronate is preferably used for hyaluronic acid to provide suitable water solubility.

Polymers in water-soluble gel formulations for topical or incisional injury treatment may be selected from the group consisting of ethylene polymers, polyoxyethylene-polyoxypropylene copolymers, oligosaccharides, proteins, polyethylene oxides, polyacrylamides Group of polymers and their derivatives or salts. It is understood that polyethylene oxide includes polyethylene glycol. The polymers used in the gel formulations for the treatment of anterior chamber injuries of the eye may be the same except that polyoxyethylene-polyoxypropylene copolymers or polyethylene oxides are not preferred. In addition, for use in the anterior chamber of the eye, biodegradable polymers are preferred, that is, they can be degraded into harmless components that can be effluxed from the anterior chamber of the eye or metabolized. In low viscosity, aqueous formulations for eye injury treatment, the gel-forming polymers may be the same as for topical and incisional injury treatment, except that polyethylene oxide is less preferred.

Ethylene polymers (also known as substituted polyethylenes) useful in the present invention may be selected from the group comprising polyacrylic acid, polymethacrylic acid, polyvinylpyrrolidone and polyvinylalcohol. Oligosaccharides useful in the present invention may be selected from the group comprising cellulose or cellulose derivatives, glycosaminoglycans, agarose, pectin, alginic acid, dextran, starch and chitosan. α-amylose with better water solubility is preferred. The glycosaminoglycan may be selected from the group comprising hyaluronic acid, chondroitin, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate and heparin . Glycosaminoglycans can be used in combination with other gel-forming polymers to enhance the treatment of wounds. The protein of the invention may be selected from the group comprising collagen, gelatin, and fibronectin. The polyacrylamide polymer may be polypropylene or polymethacrylamide polymer. Biocompatible polyacrylamide polymers are preferred.

The viscosity of gel formulations for topical and incisional lesion treatment may range from 1,000 to 12,000,000 cps at room temperature. The preferred viscosity range is 1000-2,000,000. A more preferred viscosity range is 1,000-500,000. The most preferred viscosity range is 1000-150,000 cps. The topical gel formulations of the present invention may contain 0.1-5% by weight of carboxymethylcellulose (CMC) or sodium carboxymethylcellulose (NaCMC) having a molecular weight of about 450,000-4,000,000. In a preferred embodiment, 1.5-4% by weight of CMC having a molecular weight of about 450,000-4,000,000 is present. The pH value of the CMC gel should be in the range of 4.5-8, preferably in the range of 5-7.

In another aspect, the gel formulation for topical and incisional injury treatment of the present invention may comprise 15-60% by weight of a polyoxyethylene-polyoxypropylene copolymer having an average molecular weight of about 500-50,000. In a preferred embodiment, the block copolymer accounts for 15-40% by weight and has a molecular weight in the range of 1,000-15,000. The block copolymers used in the present invention are typically Pluronics. Pluronics F88 and F127 are preferred.

In a further embodiment, the gel formulation for topical and incisional injury treatment of the present invention may comprise 0.5-10% by weight of hyaluronic acid having a molecular weight in the range of 500,000-8,000,000. In a preferred embodiment, hyaluronic acid with a molecular weight greater than 1,000,000 is contained in an amount of 1.5-6.0% by weight.

Polyacrylamide polymer can be used in the treatment of various injuries, especially the treatment of the anterior chamber of the eye. Absorbable acrylamide polymers, such as polyacrylamide, are good alternatives to carrier systems currently used in ocular treatments, such as hyaluronic acid. The molecular weight of the acrylamide polymer can be in the range of 1-13,000,000, preferably about 4-6,000,000. The weight percentage of polyacrylamide polymer in the gel can be 2-5%, preferably 3.5-4.5%. Substituted polyacrylamide polymers, such as methyl or alkyl substituted polymers, are also within the scope of this invention.

The acrylamide gel drug delivery system for use in the anterior chamber of the eye has the following characteristics: any product dissolved or degraded by the drug delivery matrix is non-toxic and does not interfere with the work of the trabecular meshwork; the gel is visually transparent; and the gel can be left in the The anterior chamber of the eye without any clinical adverse effects, such as unacceptable elevation of intraocular pressure.

It will be apparent to those skilled in the art that the desired viscosity can be achieved by varying the molecular weight and percent concentration of the polymers in the formulation. For example, low viscosity gel formulations can be achieved using low molecular weight polymers or low percentage concentrations or a combination of both. High viscosity gums can be achieved with higher molecular weights and higher percentage concentrations. Intermediate viscosities can be achieved by similarly varying molecular weight and percent concentration.

Gel formulations requiring lower viscosity than gels for topical and incisional injuries, i.e. formulations for the treatment of anterior chamber injuries of the eye and low viscosity solutions for the treatment of eye injuries, the percentage concentration of the polymer can be varied and molecular weight to achieve the desired viscosity. For example, in anterior chamber applications of the eye, the gel may comprise 1-20% by weight of a cellulosic polymer having a molecular weight in the range of 80,000-240,000. The preferred concentration range is 1-3%. Another protocol for application to the anterior chamber of the eye may include hyaluronic acid having a molecular weight of 500,000-8,000,000 at a concentration of 0.5-5%. Preferably, the concentration of hyaluronic acid is 0.5-2.0%, and the molecular weight is 2,000,000-4,000,000. The preferred viscosity range for use in the anterior chamber of the eye is 1000-100,000 cps.

The low viscosity solution may include 0.1-2.0% by weight of polyacrylic acid having a molecular weight of about 100,000-4,000,000. In a preferred embodiment, the polymer content is 0.05-0.5%. In another embodiment, the dilute viscosity solution may contain 2-40% by weight of polyoxyethylene-polyoxypropylene copolymer with an average molecular weight of 500-500,000. Preferably, the concentration is 2-20%, and the molecular weight is about 1,000-15,000. Alternatively, the dilute viscosity solution may include 1-20% cellulosic polymer having a molecular weight of about 80-240,000. The preferred concentration is in the range of 1-10%. In a further embodiment, the dilute solution may comprise 0.5-5.0% by weight hyaluronic acid having a molecular weight of about 500,000-8,000,000. The preferred concentration is 0.5-2.0%, and the molecular weight is 1,000,000-6,000,000. If the solution of dilute viscosity is to be used as eye drops, it is preferable that the viscosity is in the range of 1 to 1000 cps. If used otherwise, such as soaking bandages, then any concentration in the range of 1.0-5,000 will do.

Cellulosic polymers used in the gels of the present invention are capable of stabilizing polypeptide growth factors so that they do not lose their biological activity in aqueous solution. Cellulosic polymers used to stabilize EGF against loss of biological activity are described in US Patent No. 4,717,717. The cellulose polymer used in the present invention is a water-soluble etherified cellulose polymer, such as cellulose, hydroxyalkyl cellulose and alkyl hydroxyalkyl cellulose, such as methyl cellulose, hydroxyethyl cellulose, carboxylated Methylcellulose, hydroxypropylmethylcellulose and hydroxypropylcellulose. Derivatives of methylcellulose and hydroxyalkylcellulose such as carboxymethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose are preferred.

The stability of PDGF cellulose polymer gel formulations can be increased by adding charged chemicals such as charged amino acids or metal ions to the formulation. Suitable amino acids that may be used include lysine, arginine, histidine, aspartic acid, glutamic acid, alanine, methionine, proline, serine, asparagine and tryptophan. Aminoguanidine and protamine are also available. Suitable metal ions for use in gel formulations include zinc and magnesium. Amino acids can be used as free acids or their salts such as hydrochlorides. "Stability" as used herein refers to preventing loss of PDGF mitotic activity in the gel or increase in the amount of released PDGF protein in the gel. Thus the present invention provides a PDGF gel formulation useful for treating wounds.

The increase in the stability of PDGF in CMC gels can be accomplished by reducing the charge interaction between PDGF or by adding competing positive or negative counterions to reduce the interaction with the shortened tail of CMC. It is advantageous to add preservatives to the preparation or to sterilize the preparation by, for example, filtration or heating at a pressure of up to 1 bar applying a temperature of about 122° C. for several minutes.

The fibers of absorbent gauze may be coated with the gel formulations of the present invention to form therapeutic bandages which may be attached to wounds. Low viscosity formulations are preferred for this application. The wound treatment bandage is preferably obtained by soaking gauze with an aqueous gel solution containing a polypeptide growth factor having mitotic activity. The bandage can then be applied to the wound so that the coated fibers of the gauze come into contact with the wound, promoting cell growth to increase the speed of wound healing.

The gel of the present invention can be used in eye drops, eye rinse solutions, ointments for wound recovery, and the like. Injuries treatable with the compositions of the present invention are epithelial injuries such as eye injuries resulting from accidental or medical injury, injuries in the eye induced by corneal ulcers, radiation keratotomy, corneal transplantation, suprakeratology lens transplantation and other surgical procedures. and skin injuries such as burns, incisional injuries, wounds and ulcers at the donor site in skin grafts (skin, decubitis, stagnant veins and diabetes). Treatment of ocular injuries as used herein includes treatment of anterior chamber injuries and treatment of subconjunctival injuries. The gels of the present invention are also useful in the treatment of internal incisional injuries and internal wounds such as gastric ulcers.

In applications where the gel is used in the treatment of internal or incisional injuries, it is preferred that the gel-forming polymer is degradable. Naturally occurring polymers are generally degradable. Such as collagen, glycosaminoglycans, gelatin and starch. Cellulose cannot be degraded. Synthetic polymers such as ethylene polymers are not degradable. The degrees of biodegradability described herein are well known to those skilled in the art.

The examples given below serve to illustrate the invention. The invention is not limited by these examples, but only by the appended claims.

Example 1

carboxymethyl cellulose gel

Carboxymethylcellulose (CMC or NaCMC) gels were prepared according to the invention. Sodium carboxymethylcellulose is preferably CMC (NaCMC) 7H3SFPH of pharmaceutical grade. In a 350 liter Fryma mixing vessel the following ingredients were added to 226.2 kg of water for injection (WFI) at 80°C: 1946.3 grams of sodium chloride and 1203.5 grams of L-lysine hydrochloride. The mixture was stirred for 10 minutes. The internal homogenizer was turned on (approximately 2850 rpm) and 5776.8 grams of NaCMC were added within 5 minutes. After 10 minutes the homogenizer was turned off. The mixture was stirred at 44 rpm and cooled to 25°C. When the temperature reached 25[deg.] C., the mixture was evacuated at -0.8 bar. The mixture was then sterilized at 122°C for 20 minutes.

After sterilization, the mixture was cooled to 25°C and mixed with the following components: 11.721 liters of WFI, 377.9 grams of sodium acetate trihydrate and 15.6 grams of acetic acid. These components are aseptically added to the gel by filtration through a sterile filter. The mixture was stirred for 30 minutes. A solution of rhPDGF-B (25 g) in 2500 mL of WFI was sterile filtered and added to the gel with stirring. Then lightly wash with 250 ml WFI. The gel was mixed for 2 hours and then aseptically transferred to a stainless steel transfer container. The gel is transferred to a sealer and sealed into a sealed container. The gum had a viscosity of 2883 cps measured at 37°C.

Example 2

    Carboxymethyl cellulose gel

Carboxymethyl cellulose gel was prepared as follows:

Add 3.24 g of methyl paraben and 0.36 g of propyl paraben to 1900 g of high-purity water heated to 70-80 °C and stir until dissolved by visual observation. After the paraben was dissolved, 3.14 g of sodium acetate and 8.086 g of sodium chloride were added and the heat source removed. After dissolution, the solution was cooled to 15-30°C. To the cooled solution was added 136 µl of acetic acid and 1.73 ml of m-anisole. After thorough stirring, the pH of the buffer was measured (PH=5.63) and water was added to the final volume (2 L). The final pH of the buffer was 5.60. To 585.6 g of buffer in a 1 liter polycarbonate bottle was added 3.0 g of L-lysine hydrochloride and the mixture was stirred until visually dissolved. Using a Lightning Labmaster mixer set at 1300 rpm, 14.4 grams of Aqualon CMC, grade 7H3SFPH, was added to the buffer over 30 seconds through a modified funnel, and the gel was co-mixed for 90 minutes to yield a 2.4% CMC gel.

The gel was formulated based on the optical density (OD @ 280 nm) of the rhPDGF-B drug solution at 100 micrograms/gram rhPDGF-B/gm as follows: Add 6 grams of rhPDGF to 2.4% CMC gel by syringe -B drug (OD=10.0 mg/ml), drug was added to several different locations of the gel. The gel was mixed by hand for several minutes with a Heidolph mixer at 300 rpm for 1 hour under 0.2 micron-filtered nitrogen. Roll the gel on a roller at low speed for 1 hour. Then rhPDGF-B/CMC gel was packaged into a 15-gram capacity Teledyne layered test tube (about 10 grams/tube), and the test tube was heat-sealed with Kalix. Total yield is 55 tubes.

Example 3

    Carboxymethyl cellulose gel

Carboxymethylcellulose (CMC or NaCMC) gels were prepared according to the invention. Sodium carboxymethylcellulose is preferably CMC (NaCMC) 7H3SFPH of pharmaceutical grade. 48.6 grams of methyl paraben and 5.4 grams of propyl paraben were added to 28.4 kg of water in a 50 liter Fryma mixing vessel and heated to 80°C for 1 hour. The mixture was cooled to 30°C and the following components were added at 16 rpm: 47.1 g of sodium acetate, 242.6 g of sodium chloride and 150 g of L-lysine hydrochloride and 2.0 g of glacial acetic acid. The anchor motor was turned off, the dissolving bottle was set between 1300 and 1600 rpm, and 720 grams of CMC powder was added in less than 1 minute. The anchor motor was then started and the resulting mixture was mixed for 1 hour. Close the dissolution bottle after 6 minutes.

After mixing for 1 hour, the mixture was sterilized at 122°C for 20 minutes. After sterilization, cool to room temperature. 27 g of m-anisole were added and the resulting mixture was mixed for an additional hour. Add PDGF to the sterile gel using aseptic technique. The resulting gel was stirred for 1 hour. The gum had a viscosity of 8200 cps measured at 37°C.

Example 4

    Carboxymethyl cellulose gel

Carboxymethylcellulose (CMC or NaCMC) gels were prepared according to the invention. Pharmaceutical grade sodium carboxymethylcellulose (7H3SFPH) was used in this preparation. 247.4 kg of purified water was added to a 350 liter Turbo Emulsifier mixer. With the internal homogenizer on the highest setting (1400 rpm), the following ingredients were added to the mixer: 405 grams of methyl paraben and 45 grams of propyl paraben. The mixture was homogenized for 5 minutes, then stirred at 16 rpm, and the temperature was raised to 60°C. After the temperature was raised to 60°C, the solution was stirred for 1 hour. Cool to 30°C. The following components were added to the solution: 2020 grams of sodium chloride, 392 grams of sodium acetate trihydrate, 1250 grams of L-lysine hydrochloride, 16.25 grams of acetic acid and 225 grams of m-anisole. The resulting mixture was stirred for 15 minutes. The solution was transferred to a 600 liter tank and 6000 grams of sodium carboxymethylcellulose was added to the funnel of the Flashblend homogenizer. The liquid is pumped back into the Turbo Emulsifier mixer through the Flashblend homogenizer, and CMC is added to the buffer.

The formed gel was stirred for 2 hours. A solution of 26.05 g of rhPDGF-B in 2631 g of water was added thereto, followed by a light wash with 100 g of water. The gel was then mixed for 2 hours. Evacuate the mixing vessel, breaking the vacuum with nitrogen.

The glue was then poured into 15 gram test tubes. A final viscosity of 75812 cps, measured at 37°C, was obtained.

Example 5

        Polyacrylic Gel

Polyacrylic acid gel (Carbopol) was prepared according to the invention. Preferred grades of polyacrylic acid are known as Carbopol 934P and 940 at a concentration of 0.02-1.5%. Higher concentrations of polyacrylic acid decreased the release rate of EGF. The viscosity of polyacrylic acid is generally stable in the range of pH 6-10, preferably 6.5-7.5.

In a 4 liter beaker the following ingredients were mixed: 6.3 grams of methyl paraben, 0.7 grams of propyl paraben and 177.5 grams of mannitol in 3500 ml of water. The solution was mixed under a paddle mixer until the solids dissolved. Polyacrylic acid (17.5 g, Carbopol 940, BF Goodrich) was sieved through a 40 mesh screen and added to the solution mixing at 1000 rpm. This disperses and swells the polyacrylic acid microparticles. A 10% solution of 7.6 g of solid NaOH was added to neutralize to pH 7.0. A 900 gram portion was removed from the gel and autoclaved to obtain a sterile gel. The rest of the method takes place in the class100 area. 1.18 mg/ml (12 ml) EGF stock solution was filtered through a 0.22-micron filter membrane into a sterile test tube, washed with 5 ml of water, and filtered into the same test tube. Add the contents of the tube to the gel with a syringe. Mix the gel thoroughly with a paddle mixer to form a homogeneously dispersed EGF. Gels are placed in autoclaved containers. Press the gel out of the pressure vessel with nitrogen gas, through sterile tubing into a 10 mL syringe. Detect the activity of sample, show that every milliliter of EGF contains 15.6 microorganisms, and the sample of 10 grams does not contain microorganisms. Gels prepared had viscosities ranging from about 490,000 to about 520,000 cps. This gel formulation was used in the partial thickness skin incision injury model in pigs and guinea pigs and the gel showed accelerated and better quality wound healing in these animals.

Example 6

Pluronic Gel Formulation

Polyoxyethylene-polyoxypropylene block copolymers (Pluronic) have great potential in topical drug delivery systems due to the fact that they exhibit opposite thermal erosion behavior and that they have good drug release characteristics and low toxicity. Low molecular weight Pluronic copolymers do not form gels at any concentration in water. The minimum concentration of Pluronic F-68 to form a gel at room temperature is 50-60%. Pluronic F-68 at a concentration of 40% forms a gel at room temperature, and Pluronic F-108 forms a gel at a concentration of 30% at room temperature. Pluronic F-127 forms a gel at a concentration of only 20% in water at 25°C. Pluronic F-68, F-88, F-108, F-127 are available as pharmaceutical dosage forms for controlled release administration of EGF to burns and other donor sites. The gel should be isotonic, preferably with a pH in the range 6-8, more preferably 6.5-7.5.

An interesting property of Pluronic gels is their ability to gel as a function of temperature and polymer concentration. Gels are formed when Pluronic solutions are heated. Therefore, the gel is a low-viscosity aqueous solution at room temperature. When it contacts with the human body and heats up the body temperature, the viscosity increases and the solution becomes a gel. EGF can be combined with Pluronic in a solution state and applied to the injured area. At this point, coagulation, which effectively reduces the release of EGF to the lesion, occurs. Prolong the contact time between EGF and the epithelium of the wounded area. Gels can be applied in liquid form or combined with dressings (soaked in liquid) to provide mechanical support. The advantages of using Pluronic gels include that these gels can be sterilized by filtration and that the wound can be in contact with EGF for a long time.

Pluronic F-127 with EGF was prepared by mixing the following: 1.8 grams of sodium phosphate monohydrate, 5.48 grams of disodium phosphate heptahydrate, and 46.9 grams of mannitol in 1,000 milliliters of distilled water. The pH was adjusted to 7.0, and the solution was cooled to 4°C. Pluronic F-127 was gradually added to the cooling solution with mixing in a paddle mixer. The solution was mixed for 30 minutes and left overnight at 4°C. EGF in aqueous solution can be added to the solution before the gel is prepared or mixed after Pluronic F-127 is dissolved in the solution. To obtain an EGF concentration of 100 mg/ml, 1.812 ml of EGF solution (1.38 mg/ml) was added to 23.188 g of 20% Pluronic F-127 gel. The solution is very liquid-like. The viscosity of the solution increased as it was heated to 35°C, as shown in Table 1.

Table 1

Temperature °C Viscosity (cps,0.5rpm)

0-16 Not detected

18 4,000

19 250,000

21 500,000

28 655,000

30 685,000

37 650,000

Pluronic formulations with viscosities of 1,000,000 and 12,000,000 were also prepared. The release kinetics of the formulation were measured (11.5 x 106 cps) and it was noted that 85% of the EGF was released from the formulation within 1 hour.

Example 7

                                                                 

A number of HPMC gel formulations were prepared. Gels are made from very low to high molecular weight HPMC. The preferred molecular weight range is 80,000-240,000. A very low molecular weight polymer (Methocel E15LV) required as much as 10-20% HPMC to form a gel. Very high molecular weight polymer (Methocel K100M), only 1-3% solution is needed to form a gel. Gels were prepared at different grades and at different concentrations to study the release kinetics. The pH of each gel was adjusted to 7.2. The release rate of EGF is proportional to the viscosity of the soluble gum.

Add 0.83 g of sodium phosphate monohydrate, 7.24 g of disodium phosphate heptahydrate, 6.22 g of NaCl, and 500 mL of sterile water for rinsing into a 1,500 mL beaker. The mixture was stirred magnetically to dissolve the solids and the pH was adjusted to 7.2. The solution was heated to 80° C. with stirring, and 30.0 g of HPMC (Methocel K100M; Dow) was added through a 40-mesh sieve. Remove from heat and stir for another 10 minutes. The remaining 500 g of water was added in the form of ice. As the mixture becomes thicker, stir by hand. Cool to room temperature and then to 4°C overnight. A 130 g portion was removed and mixed with 13.4 ml of a sterile solution of 1.12 mg/ml EGF under the action of a paddle mixer to obtain an EGF concentration of 104 mg/ml.

The prepared gel has a viscosity of 54,000-950,000 cps at room temperature. The release of EGF from different HPMC gel formulations prepared is listed in Table 2.

Table 2

Sample Brookfield viscosity (CPS) Release of EGF at 25°C 37°C 2085-91-1 E4M 4% 112×10 ³ 102×10 ³ 75% in 5 hours 2085-92-2 E4M 5% 274×10 ³ 300×10 ³ 75% in 5 hours 2085-92-2 E4M 6% 652×10 ³ 946×10 ³ 50% in 5 hours 2085-91-2 E4M 4% 112×10 ³ 286×10 ³ 75% in 5 hours 2085-93 K15M 3% 102×10 ³ 70×10 ³ 75% in 5 hours 2085-91-3 E4M 4% 92×10 ³ 54×10 ³ 75% in 5 hours

Example 8

    Hyaluronic Acid Gel Preparation

Hyaluronic acid (HA) is one of the mucopolysaccharides with a repeating linear structure comprising N-acetylglucosamine and glucuronodisaccharide units. HA exists in nature, microorganisms, skin and connective tissues of humans and other animals. Depending on the source, preparation method and detection method, the molecular weight of HA is in the range of 50,000-8,000,000. High-viscosity HA solutions have lubricity and a good moisture-enhancing effect. HA is found in synovial fluid in joints, in the vitreous of the eyeball, in the umbilical cord, skin, blood vessels and cartilage. It is particularly useful as a lubricant and shock-absorbing agent, probably due to its good ability to retain water and its affinity for binding to proteins. It is safe to use in the human body. Therefore, it can be used in the treatment of internal injuries such as joints or the anterior chamber of the eye. A 1% solution of sodium hyaluronate (molecular weight 4,000,000; MedChem) was formulated with EGF to give a concentration of 100 mg/ml. The viscosity of a 1% HA solution is 44,000 cps. HA/EGF formulations prepared according to the present invention have been shown to stimulate endothelial re-healing in the anterior chamber of the eye.

Example 9

Kinetics of EGF Release from Pharmaceutical Dosage Forms

The effectiveness of various dosage forms in the sustained release of EGF in the in vitro diffusion cell system was evaluated, and the values of T ₂₅ and T ₅₀ were determined. The release of EGF is the result of its diffusion from the gel and dissolution of the gel matrix. Taking these two processes as possible mechanisms for the bioavailability of EGF in vivo, HPMC gel had the highest values for sustained release of EGF, with _T25 and _T50 values of 1.2 and 5.9, respectively. The results show that the molecular structure of the polymer is more important than the polymer concentration in prolonging the T value. Gels prepared in a salt-free matrix (distilled water) under standing conditions yielded low T-values. This may be a result of the combination of faster dissolution and higher viscosity of the salt-free gel. Accordingly, the gels of the invention are preferably not salt-free. It is predicted that the T value of the gel of the present invention can be improved by polymer modification, such as introducing hydrophobic or hydrophilic side chains, ion-pair groups, metal ions, cross-linking agents, and the affinity group of EGF to control EGF from the obtained Release in product form.

Table 3 summarizes the release kinetics of EGF from pharmaceutical dosage forms. Different letters after HPMC in the table represent the percentage of substitution in the polymer. For example, K=2208 or 22% methylation and 8% hydroxypropyl substitution; F=2906; and E=2910. The value after the letter (that is, the value 100 after K) refers to the viscosity of a 2% solution in water, and the unit is thousand cps. AQ refers to gels prepared in salt-free solution. All other gels were prepared in phosphate buffered saline (PBS), pH about 7.0. The unit of T value is hour.

Table 3 Summary of Kinetic Data for EGF Release by Dosage Form Viscosity Polymer CDS T ₂₅ T ₅₀ HPMC K100M (mw 240,000) 1% - 0.0854 1.000

2% 287×10 ³ 0.4687 1.9172

3.5% 116×10 ⁶ 1.2270 5.8528

4.0% - 0.8536 4.1386

5.0% 3.07×10 ⁶ 0.8807 3.5808HPMC K-15M (mw 120,000) 3% 122×10 ³ 0.857 2.0635

4AQ％ 331×10 ³ 0.2727 1.6900HPMC K-4M(mw 86,000) 4％ 96×10 ³ 1.0476 2.6349HPMC F-4M(mw 86,000) 4％ 122×10 ³ 0.7619 1.8730HPMC E-4M(mw0) 4％ 128×10 ³ 1.0159 2.2657

5% 312×20 ³ 0.8615 1.8462HPMC E-4M (mw 86,000) 5AQ% 240×10 ³ 0.3211 1.6044

6AQ％ 680×10 ³ 0.6944 3.0040Carbopol 934P(mw 3×10 ⁶ ) 0.5％ 494×10 ³ 0.2727 0.7300Pluronic F-127(mw 12,000) 20％ 1.1×10 ⁶ 0.1936 0.3548

Example 10

     Polyacrylamide Gel Preparation

The polyacrylamide/EGF gel formulation was prepared with polyacrylamide Cyanamer N-300 and Cyanamer N-300 LMW (both available from Cyanamid, USA). Cyanamer N-300 has a molecular weight of about 5-6 million and Cyanamer N-300 LMW has a molecular weight of about 13 million.

Polyacrylamide gels of the following compositions were prepared by adding polyacrylamide polymers to premixed saline solutions. These gels were then used to measure the release of EGF.

                                                                                  

2085-140A 2085-140BCyanamer N-300 4.0 -Cyanamer N-300 LMW - 4.0 Sodium Chloride 0.049 0.049 Potassium Chloride 0.075 0.075 Calcium Chloride 0.048 0.048 Magnesium Chloride 0.080 0.080 Sodium Acetate 0.890 0.890 Anhydrous Sodium Citrate 0.170 Sterile Water 94.688 94.688 viscosity, cps. 552×10 ³ 132×10 ³

To 1.809 g of Cyanamer N-300 (2085-140A) polyacrylamide was added 72.4 ml of a mixture ^of125I -EGF and EGF and mixed in two 3 liter syringes. 300-400 mg of this gel was placed at the donor site of the Franz diffusing cells. At predetermined time intervals, 50 ml aliquots of the receiving buffer are counted on a gamma counter. The receiving buffer contained 3.5 ml of PBS, including 0.4% BSA (bovine serum albumin) 0.02% sodium azide. Similarly, 44.5 ml of a mixture ^of125I -EGF and EGF was added to 1.11 g of Cyanamer N-300 LMW (2085-140B) polyacrylamide, and the release of EGF was measured.

Another polyacrylamide gel (2085-138C) was prepared with the following formulation.

Table 5 Components 2085-138CCyanamer N-300 7.0g sodium ethylmercury thiosalicylate 0.2g sterile water 192.8g viscosity 258×10 ³ pH 7.54

A gel containing 10 mg/ml EGF was prepared using 2085-138C gel. Weigh 5 g of 2085-138C gel into 8 serum bottles and add 50 mg of 1 mg/ml EGF (1.41 mg/ml for protein test).

EGF is coated on the wound dressing to obtain better release characteristics. The wound dressing was a gel film composed of polyacrylamide/agarose (Geliperm, Geistlich-Oharma; Wolhusen, Switzerland). The dressing was soaked with EGF aqueous solution, and the dressing was coated with EGF. Approximately 70% of the EGF was released from the dressing within 24 hours.

    Stable PDGF Cellulose Polymer Gel Formulation

Carboxymethylcellulose (CMC) is prepared according to the invention. The CMC used in the study was pharmaceutical grade 2.4% sodium carboxymethylcellulose (Aqualon Co., Wilmington, DE) with a molecular weight in the range of 900,000-2,000,000 Daltons. CMC glue was formulated with rhPDGF-BB (Chiron Corporation, Emeryville, California). It was found that the stability of PDGF cellulose polymer gel formulations can be increased by adding charged chemicals to the formulation, such as charged amino acids or metal ions. "Stability" as used herein refers to preventing loss of PDGF mitotic activity in the gel or increase in the amount of released PDGF protein in the gel. Thus the present invention provides a PDGF gel formulation useful for treating wounds.

The in vivo wound healing efficacy of the formulation was determined in a guinea pig partial thick skin excisional injury model.

In the guinea pig partial thickness skin excision injury model, two partial thickness skin excision lesions were made on each guinea pig (4-8 animals per group) with a dermatome. For the first 5 days (0-4 days), 3 x 1 cm wounds (typically about 0.4-0.8 mm deep) were treated once daily with 0.3 ml glue, covered with sterile absorbent pads. The pads are covered with an occlusive dressing and each treatment is covered with a protective bandage. At 7 days, the injured area was collected for histological evaluation. The average thickness of granulation tissue was determined by projecting Gomori trichrome-stained tissue sections on a computerized digital planimeter at 50X magnification. Thickness was obtained by dividing the area by the length following the standard length of granulation tissue in the lesion. Three tissue areas were measured per wound and the values obtained from the two wounds per guinea pig were averaged to obtain values for each animal.

In this model, PDGF at concentrations of 10-300 μg/g in CMC preparations induces a 2-3 fold (or greater) increase in the thickness of granulation tissue in the lesion. Localized granulation tissue is newly formed connective tissue and blood vessels that are a major component of wound healing. Granulation tissue physically fills the lesion. In addition, granulation tissue provides the rich blood flow needed for the epithelium to grow over the injured surface. Therefore, granulation tissue is an essential component in the treatment of wounded areas.

The table shows 1) the potency of PDGF in CMC glue, 2) the lack of lysine potency on the impact of PDGF potency in the newly prepared batch (table 6), 3) lysine stable formulation potency retained for 30 months (stored in 2 -8°C) (Table 7). And 4) Retention of PDGF potency in sterile non-preserved (with lysine) CMC preparations (Table 8).

N

Thickness (mm) (animal)

Mean ± SEM Original CMC 63.3 ± 15.9 PDGF in 7CMC (30 μg/g) 161.9 ± 25.4 ^* 7CMC + 0.1% lysine 64.4 ± 8.9 PDGF in 7CMC (30 μg/g) + 0.1% lysine 162.6 ±23.5 ^* 8CMC+0.5% Lysine 59.9±5.6 PDGF in 8CMC (30 μg/g)+0.5% Lysine 144.6±21.8 ^* 7 ^* Significantly different from vehicle control (p<0.05)

The data indicate that recombinant human thrombopoietic growth factor-BB in CMC gel was effective (here 2.5-fold) in increasing the number of granulation tissue in experimental guinea pig lesions. The data also indicated that up to 0.5% lysine did not affect the potency of PDGF in CMC gels.

Table 7 Effect of PDGF in Lysine Stabilized CMC on Granulation Tissue Thickness in Guinea Pig Partial Thickness Wounds Treatment Granulation Tissue N Thickness (mm) (animal) mean ± SEMCMC ^* 81.4 ± 15.5 311.3 ± in 630 monthly batches of CMC 32.4 ^** 6PDGF (30 μg/g) 217.3 ± 22.3 ^** 6PDGF (30 μg/g) in freshly prepared batches of CMC

^* All formulations containing 0.5% lysine

^** Different from vehicle control group (p<0.05)

These formulations containing 0.5% lysine maintained their potency in promoting test Dutch pork sprout tissue formation for at least 30 months.

Table 8

Effect of PDGF in sterile unpreserved CMC preparations on the thickness of granulation tissue in partial-thickness wounds of guinea pigs Treatment of granulation tissue N

Thickness (mm) (animal)

Mean ± SEM Aseptic CMC ^** Unpreserved 150.1 ± 48.5 652.6 ± 58.6 ^*** 4PDGF (100 µg/g) in sterile unpreserved CMC 635.1 ± 73.5 ^*** 4PDGF ( 100 μg/g) 752.2±79.5 ^*** 4PDGF in sterile unpreserved CMC (100 μg/g)

^* This model was done in a different laboratory with deeper wounds than in the lysine assay, resulting in a higher thickness of granulation tissue in the vehicle control.

^** All formulations containing 0.5% lysine.

^*** Different from vehicle control (p<0.05)

These data indicate that sterile, unpreserved PDGF/CMC (with 0.5% lysine) has the potential to promote granulation tissue formation in wounded areas.

The mechanism by which charged chemicals can stabilize PDGF/cellulose polymer formulations is unclear. Without wishing to be bound by theory, the stabilizing effect may be due to competition of charged species with PDGF lysine for the reducing end of the cellulose polymer and/or by reducing the interaction between the free carboxylate of the cellulose polymer and PDGF lysine. Charge interaction occurs. Charge interactions between PDGF and cellulose polymers such as CMC have been demonstrated in our laboratory. Charged chemicals that can compete for positive and negative charges can reduce the charge interaction between PDGF and cellulose polymers, thereby increasing the stability of PDGF. The ability of the positive counterion to increase the recovery of PDGF from the CMC gel was tested.

Answering the question of PDGF-CMC stability first requires an understanding of how PDGF is inactivated in the presence of CMC. One possible mechanism for the loss of activity is the adsorption and entrapment of PDGF in CMC. The nature of this adsorption involves the entrapment of the highly positive PDGF B dimer (comprising 22 arginine and 14 lysine residues) with a negative CMC matrix containing a large number of free carboxyl groups. Our studies show that in the presence of little or no counterion to offset the charge interaction, the mitotic activity of PDGF in CMC is reduced, and the protein release is reduced.

The stability of PDGF in CMC gels can be increased by adding competitive positive or negative counterions to reduce the charge interaction between PDGF or reduce its interaction with the reducing end of CMC. In one experiment, lysine (0.5% w/w) increased the stability of PDGF in CMC gels by more than 50%. Any compound that reduces or competes for the PDGF-CMC charge interaction or its interaction with the reducing end of CMC increases PDGF stability in CMC gels. It should be understood that the charged chemical species in the formulations of the present invention are initially added to the formulation in the form of a salt. The salt then dissociates in the gel's aqueous environment to produce differently charged chemicals. The term "charged chemical" as used herein includes pharmaceutically acceptable forms of: salts, such as zinc chloride and magnesium chloride; buffers, such as mono/diethanolamine, fumaric acid, malic acid, potassium citrate and sodium gluconate; amino acids such as lysine, arginine, histidine, aspartic acid, glutamic acid, alanine, methionine, proline, serine, asparagine, tryptophan; Aminoguanidine and protamine; ionic surfactants, such as oleic acid and oleth5; and synthetic polycationic or anionic polymers, such as polyamino acids. The term "pharmaceutically acceptable" as used herein means that the material can be used in the treatment of humans or other mammals without causing side effects such as toxicity, herpes or albinism of mucous membrane or skin tissue.

In addition to the tests listed below, 0.5% aminoguanidine also increased the recovery of PDGF from the cellulose polymer gel. Due to its stronger nucleophilicity, aminoguanidine is a better competitor than lysine in preventing non-enzymatic glycosylation of proteins.

The present invention is intended to include formulations comprising cellulosic polymers, or other potentially reducing-terminated polymers, and pharmaceutically acceptable nucleophilic counterions, such as lysine or aminoguanidine.

Test #1

The samples used were rhPDGF-B mixed with CMC placebo for 0.5 hours and rhPDGF-BB in CMC mixed for 60 days at 4°C. Table 9 Effect of zinc ion concentration on PDGF recovery. The 0-day samples mixed with the same zinc ion concentration showed a higher recovery percentage than the 60-day samples. These data suggest that PDGF binds ionically to CMC, and that over time, this binding cross-links CMC, requiring more zinc ions for PDGF release.

PDGF recovery was determined on a 300 Angstrom wide-pore C4 reverse phase HPLC column using a gradient of 10-70% acetonitrile containing 0.1% trifluoroacetic acid.

Table 9

RHPDGF resumes % Moore concentration, zinc 0.075 84 710.15 88 810.3 97 90 is 0 days.

Table 10 shows the effect of other charged compounds on the recovery of PDGF in CMC gels. 0.5 g of PDGF samples mixed for 60 days were dissolved in 10 ml of water containing 0.01% BSA (used as a control), and the listed compounds were added to each sample.

Table 10 sample %PDGF recovery A control 50B 0.2M NaCl (mono+) 42C 0.2M ZnCl ₂ (di++) 64D 0.2M CaCl ₂ (di++) 63E 0.2M MgCl ₂ (di++) 69F 0.5% glycine (+, lysine) 64G 0.5 glycine (zwitterion) 50

These data indicated that the more electropositive Zn, Ca, Mg and lysine could reduce the charge interaction between PDGF-CMC. They also suggest that PDGF may become entangled or trapped in the CMC matrix.

Test #2

This set of experiments was designed to examine the effect of different amino acids on PDGF restoration in CMC. The sample used is 100 micrograms of PDGF/gram of CMC, pH 6.0, buffer solution 0.13M NaCl, plus 0.5% amino acid. Samples were incubated in polypropylene tubes at 46°C for 2 days and then analyzed on reverse-phase HPLC as before.

Table 11 presents the relative evaluation data of the effect of polar and non-polar amino acids on PDGF recovery in CMC. Amino acids with higher charges (lysine, aspartic acid and glutamic acid) had the greatest recovery of PDGF. At pH 7.0, all amino acids are zwitterions. Therefore, depending on the charge of the R-groups, they will give higher recovery. The higher the charge of the R-group, (eg lysine), the better the stabilizing effect.

             Table 11 Effects of different types of amino acids on the recovery of PDGF in CMC RECOVERY IN CMC

Compared with zero -time control group samples, a recovery of % control, zero 100.0cmc+no AA 37.3 non -polar side chain glycine 43.4 alanine 37.5 eggine 40.8 proline 40.2 non -charge side chain linoleine 48.7 winter illegal 39.9, 39.9, Tyline 45.5 chromine 42.0 ion -type polar side chain lysine PK9 64.0 days cashine PK4 70.4 glutamate PK4 58.2

The effect of other different charged compounds on PDGF recovery in CMC was also evaluated and the data are presented in Table 12. 100 microgram PDGF/gram CMC was used in these experiments. The data showed that when the NaCl concentration increased from 0M to 1.13M, the recovery percentage of PDGF increased from 32% to 96%. 0.1MMg++ gave 94.8% recovery.

Table 12 Effect of Different Charged Compounds on Recovery of PDGF in CMC Samples Recovery Percentage

Take zero time as the reference, zero time 100.0CMC+0.0M NaCl (prepared with water only) 32.1CMC+0.0M NaCl (prepared only with buffer) 27.6CMC+0.13M NaCl 65.9CMC+0.33M NaCl 82.8CMC+0.63M NaCl 90.7CMC+0.94M NaCl 93.8CMC+1.13M NaCl 96.8CMC+0.0M NaCl+MgCl ₂ 0.1M 94.8CMC+0.13M NaCl+Gly 0.5% 76.7CMC+0.13M NaCl+Lys 0.5% 85.5 Test #3

The mitogenic activity of rhPDGF with and without lysine (30 μg/g) for one month is listed in Table 13. The data were measured by the method of thymidine uptake in fibroblasts, expressed as the content of rhPDGF measured. The data indicate that the presence of lysine in the formulation enhanced the mitogenic activity of the formulation.

Table 13RHPDGF μg/G does not include lysine (n = 10) lysine (n = 4) 25 ℃ 16.8 +/- 4 31.2 +/- 730 ℃ 10.4 +/- 27.0 +/- 0.5 0.5

    rhPDGF-BB cellulose polymer preparation

The cellulose gel formulation containing rhPDGF-BB can be prepared according to generally accepted formulation techniques. Usually, preparing an internal mixture of the desired components is all that is required. "Internal mixture" means that the ingredients of the composition are all mixed homogeneously such that none of the components are on the surface.

The compositions of the present invention include a "wound therapeutically effective amount" of PDGF, ie an amount sufficient to accelerate the rate of wound healing. As is well known in the medical arts, an effective amount of a pharmaceutical formulation will vary with the particular agent employed, the conditions at which it will be treated and the subject being treated. Therefore, an effective dose of each therapeutic agent cannot be specifically determined. A therapeutically effective dose of PDGF is therefore an amount of PDGF sufficient for the composition of the invention to provide sufficient PDGF on or in the treated organism for the desired period of time, usually less than the amount normally used. A typical composition of the present invention contains about 1.0 micrograms to about 1000 micrograms of PDGF in 1 gram. Preferably, the compositions of the present invention contain from about 1 microgram to about 300 micrograms of PDGF per gram of formulation.

Additional ingredients such as buffers, preservatives, tonicity regulators, antioxidants, other polymers (for, eg, viscosity adjustment or extenders) and excipients may also be used in the compositions of the present invention. Examples of these other materials include phosphate, citrate, or borate buffers; thimerosal, sorbic acid, methyl or propyl paraben, m-cresol, and chlorobutanol preservatives; tonicity-adjusting chlorine sodium and/or sugar; polymers such as polyvinyl alcohol, polyacrylic acid and polyvinylpyrrolidone; and excipients such as mannitol, lactose, sucrose, ethylenediamine tetraacetate, etc.

Table 14 lists typical cellulose polymer gel formulations containing rhPDGF-BB. The viscosity of this formulation is in the range of 1000-150,000 cps at room temperature.

Table 14 The concentration range RHPDGF-BB 1.0-1,000 mg per gram of glue cellulose polymer 1.5-3.0 % (W/W) Chemicals with charge 0.1-3.0 % (W/W) preservatives 0.15-0.25 % (W /w) Special formulations containing rhPDGF-B listed in Tables 15, 16 and 17

Table 15 Ingredients Quantity %(w/w) pure water 100g 96.02

Amount g/100g

Pure water hydroxyl -moles of 0.1620g 0.16 hydroxybenzoate 0.0180g 0.02 sodium trifle 0.1570 g 0.15 lysine hydrochloride 0.5000g 0.48 sodium 0.8086g 0.0900 g 0.09 methalium 0.0065 g 0.01 sodium carboxymethylcellulose 2.4000g 2.30

Amount g/100g

Gum rhPDGF-B (Bulk Stock sol’n 0.0036g

Table 16 Composition Amount/250kg Batch Amount/g Glycolic Acid 15.6g 0.06mg Sodium Chloride 1946.3g 7.70mg Sodium Acetate Trihydrate 377.9g 1.51mgL-Lysine Hydrochloride 1203.5g 4.81mg Water for Injection 240700.0g ^* 962.80mg Carboxylic Acid Sodium Methylcellulose 5776.8g 23.11mgrhPDGF-BB (bulk drug) 25.0g 0.10mg ^* Total WF1 includes 2500g in liquid drug.

Table 17 ingredients G/100g Gel Rhpdgf-BB 0.01 Cryptomymethyl Cell Crystone Sodium 2.40 Sodium 0.8086 Sodium Sodium 0.1570 Implitate 0.0065 hydroxyl-hydroxyticate 0.1620 propionate 0.0180 phenol-0.0900L- Lysine Hydrochloride 0.5000 Water for Injection 100.00

The invention is described herein with reference to some preferred versions and embodiments. It will be obvious to those skilled in the art that the invention may vary widely. In particular, one skilled in the art can vary the molecular weight and percentages of different polymers to achieve the desired viscosity. Those skilled in the art can also substitute different polymers or growth factors for those listed here in the present invention. Since variations will be apparent to those skilled in the art, the invention is not limited by what is set forth herein but only by the claims.

Claims (26)

1. A pharmaceutical composition comprising: a) platelet-forming growth factor (PDGF) in an effective wound treatment amount; b) pharmaceutically acceptable cellulosic polymers; and c) pharmaceutically acceptable charged chemical substances selected from the group consisting of lysine, arginine, histidine, protamine, alanine, methionine, proline, serine, asparagine, tryptophan, The group of aminoguanidine, zinc and magnesium, wherein the composition is an aqueous gel having a viscosity in the range of about 1000-500,000 cps at room temperature. 2. The composition of claim 1, wherein PDGF is a homodimer of PDGF-B. 3. The composition of claim 1, wherein the cellulosic polymer is selected from the group consisting of carboxymethylcellulose, hydroxypropylmethylcellulose and methylcellulose. 4. The composition of claim 1, wherein the concentration of PDGF is in the range of about 1.0 μ/gram to about 1,000 μ/gram. 5. The composition of claim 1, wherein the concentration of cellulosic polymer is about 1.5% (W/W) to 3% (W/W), and the molecular weight of the polymer is about 450,000-4,000,000. 6. The composition of claim 1, wherein the concentration of the charged species is in the range of about 0.1% (W/W) to 3.0% (W/W). 7. The composition of claim 1, wherein the viscosity is in the range of 50,000-150,000. 8. The composition of claim 1, further comprising a preservative. 9. The composition of claim 8, wherein the preservative is one or more selected from the group consisting of methylparaben, propylparaben and m-cresol. 10. A method for treating an injury in a patient comprising contacting the injury with the composition of claim 1. 11. A pharmaceutical composition comprising: a) a human PDGF-B homodimer in an effective wound treatment amount; b) Carboxymethylcellulose (CMC); and c) Lysine, wherein the composition is an aqueous gel having a viscosity in the range of about 1000-500,000 cps at room temperature. 12. The composition of claim 11, wherein the concentration of the cellulosic polymer is about 1.5% (W/W) to 3.0% (W/W), and the molecular weight of the polymer is about 450,000-4,000,000. 13. The composition of claim 11, wherein the concentration of lysine is in the range of about 0.1% (W/W) to 3.0% (W/W). 14. The composition of claim 11, wherein the viscosity is in the range of 1000-150,000. 15. The composition of claim 11, further comprising a preservative. 16. The composition of claim 15, wherein the preservative is selected from one or more of the group consisting of methylparaben, propylparaben and m-cresol. 17. A method for treating an injury in a patient comprising contacting the injury with the composition of claim 11. 18. A pharmaceutical composition comprising: a) platelet-forming growth factor (PDGF) in an effective wound treatment amount; b) cellulosic polymers having a pharmaceutically acceptable concentration in the range of about 1.5% (W/W) to 3% (W/W) and a molecular weight in the range of about 450,000-4,000,000; and c) a pharmaceutically acceptable positively charged chemical substance selected from the group comprising positively charged amino acids, positively charged polyamino acids and positively charged metal ions at a concentration of about 0.1% (W/W) to 3.0% (W/W), wherein the composition is an aqueous gel with a viscosity in the range of about 1000-500,000 cps at room temperature. 19. The composition of claim 18, wherein the viscosity is in the range of about 1000-150,000 cps. 20. The composition of claim 18, further comprising a preservative. twenty one. The composition of claim 20, wherein the preservative is selected from one or more of the group consisting of methylparaben, propylparaben and m-cresol. twenty two. The formulation of claim 18, wherein the charged chemical substance is selected from the group consisting of lysine, arginine, histidine, protamine, alanine, methionine, proline, serine, asparagine, tryptophan , the group of aminoguanidine, zinc and magnesium. twenty three. The pharmaceutical composition of claim 15, comprising (g/100 gram glue) 0.01 gram rhPDGF-B; 2.40 gram carmellose sodium; 0.81 gram sodium chloride; 0.157 gram sodium acetate trihydrate; 0.0065 gram glacial acetic acid; gram of methyl paraben; 0.018 gram of propylparaben; 0.09 gram of m-cresol; 0.5 gram of L-lysine hydrochloride; within range. twenty four. The pharmaceutical composition of claim 11, comprising (grams/100 grams of glue) 0.1 gram of rhPDGF-B; 23.103 grams of sodium carboxymethylcellulose; 7.784 grams of sodium chloride; 1.511 grams of sodium acetate trihydrate; 0.0624 grams of glacial acetic acid; grams of L-lysine hydrochloride; and 962.63 grams of water with a viscosity in the range of about 1000 to about 15,000 cps at 37°C. 25． A method of treating an injury in a patient comprising contacting the injury with the composition of claim 22. 26． A method of treating an injury in a patient comprising contacting the injury with the composition of claim 23.